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Team:Groningen/Template/MODULE/Notebook/protocols/transformationlactococcus
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| - | An overnight culture of <i>L. lactis</i> is made in SMGG. The following day, 10 ml of the overnight culture is pipetted in 100 ml SMGG and grown at 30 °C | + | An overnight culture of <i>L. lactis</i> is made in SMGG. The following day, 10 ml of the overnight culture is pipetted in 100 ml SMGG and grown at 30 °C until an OD<sub>600</sub> of 0.2 - 0.7 is reached. The cells are then three times washed with 50 ml ice cold wash buffer (0.5 M sucrose and 10% glycerol). The cells are then resuspended in 1 ml wash buffer and can be used directly or stored at -80 °C. |
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Revision as of 00:01, 18 October 2014
Transformation of Lactococcus lactis
Preparing electrocompetent L. lactis
An overnight culture of L. lactis is made in SMGG. The following day, 10 ml of the overnight culture is pipetted in 100 ml SMGG and grown at 30 °C until an OD600 of 0.2 - 0.7 is reached. The cells are then three times washed with 50 ml ice cold wash buffer (0.5 M sucrose and 10% glycerol). The cells are then resuspended in 1 ml wash buffer and can be used directly or stored at -80 °C.
Electroporation
For the electroporation 1 μl of the DNA is added to 40 μl cells in an ice cold cuvette. The cuvette is then electroporated at 2.5 kV, 25 μF and 200 &Ohm;. Then, 1 ml SMG17MC is added and the cells are incubated at 30 °C for two hours. The cells can then be plated on GSM17-agar plates.
